NOTE: End date changed to 7/31/2014 per NSSC information (Ed., 4/10/14)

NOTE: End date changed to 4/30/2014 per NSSC information (Ed., 1/22/14)

In our last project, we established that X-ray, 56Fe (iron ion), and proton irradiation of blood vessel cells increase adhesiveness of the vessel wall, and that X-rays and 56Fe accelerate development of atherosclerosis in a mouse model (results of proton experiments are pending). The molecular mechanism for this, however, is not yet known. In addition, it remains to be determined how fractionation of doses and irradiation of other tissues affect the dose dependence of both cell adhesion and development of atherosclerosis.

With the hypothesis that radiation in general and cosmic radiation in particular directly alter the adhesive properties of vascular endothelium, and resultant vascular inflammation accelerates atherosclerosis, we propose to systematically investigate mechanisms of radiation effects on vascular cells, using both isolated cells and whole mice, to better predict risk and to provide the basis to develop possible future countermeasures. Our specific aims are:

Aim 1: Determine whether atherogenic effects of radiation are limited to local effects on vascular endothelium, or if other systems contribute to disease progression and/or modify dose dependence.

Aim 2: Determine the molecular mechanism of acute activation of leukocyte-endothelial cell adhesion in response to radiation.

Aim 3: Determine how fractionation of doses affects dose-dependence of progression rates, latency periods, and surrogate endpoints.

The risk from accidental exposure is similar. For example, atomic bomb survivors have an increased incidence of coronary artery disease and stroke. Risk for cardiovascular disease after radiation exposure at Chernobyl was increased for those who were exposed to less than 1 Gy. Even radiation technologists in the 1950s (when shielding was less rigorous) had an increased risk of death from cardiovascular disease, demonstrating that repeated exposure at low doses results in significant risk years later. Currently, the principal strategy for reducing risk is avoidance of exposure.

Completion of our specific aims will advance the knowledge of the molecular mechanisms of radiation-induced atherosclerosis, enabling better prediction of cardiovascular risk from exposure, facilitating early detection through the use of surrogate biomarkers, and pointing the way toward potential countermeasures to mitigate the cardiovascular consequences of radiation exposure, both in space and on Earth.

The overall goal of this project was to determine the effect of radiation, especially cosmic radiation, on atherosclerosis, the disease that results in heart attacks and strokes. This could help to estimate the risk for astronauts on deep-space missions, such as a trip to Mars. This project consisted of two arms. The cell component, using cultured human aortic endothelial cells (HAECs) was designed to elucidate the mechanism by which absorption of radiation, either photons or ions, causes pro-atherogenic changes in the vascular endothelium. The animal component, which used development of atherosclerotic plaques as an endpoint, was to determine the eventual consequences of these changes for development of cardiovascular disease, and to work toward identification of early endpoints that predict later consequences.

ApoE -/- mice were used in the animal component of this project because they already had a well-characterized response to x-ray radiation, especially with respect to atherosclerosis. Previous studies had established that this mouse model reproduces the pathology seen in humans after radiation exposure. Moreover, these mice mimic an adult human population in that they unavoidably develop some degree of asymptomatic atherosclerotic disease spontaneously with age.

In this project, we made progress on three CPR questions, as detailed below.

Degen-1, “How can tissue specific risk models be developed for the major degenerative tissue risks, including heart, circulatory, endocrine, digestive, lens and other tissue systems in order to estimate GCR (galactic cosmic radiation) and SPE (solar particle event) risks for degenerative diseases?”

The tissue of interest for this project is the vascular system, especially the major vessels of the chest and neck, which are the major sites of clinically significant atherosclerosis.

To address Degen-1, we established that 56Fe (i.e., iron ions, a particularly damaging component of cosmic radiation) targeted to the aorta and carotid arteries has similar effects as x-rays, but at a lower dose. That is, while 8-14 Gy x-rays have been shown to exacerbate atherosclerosis in this mouse model, 56Fe has significant effects at as little as 2 Gy. The fact that 56Fe can produce effects at lower doses is not surprising, since particle radiation can be particularly damaging. In radiobiology, the relative biological effect (RBE) is defined as the ratio of absorbed dose of a radiation in question to the absorbed dose of a reference radiation (usually gamma- or x-rays) required to produce the same biological effect in a particular tissue. Based on this definition, the RBE of approximately 4-7 for 56Fe ion-induced atherosclerosis (signifying that iron ion causes similar adverse cardiovascular effects at a 4-7 fold lower dose than x-rays) is consistent with those associated with other effects.

It is known from human epidemiologic data that x-ray or gamma-ray doses as low as 1 Gy can increase the risk of cardiovascular disease, suggesting that humans are 8-14 times more sensitive to the pro-atherogenic effects of high-energy photon radiation as apo-E -/- mice. Assuming an RBE of 4-7 for humans as well as mice, the prediction would be that 56Fe as low as 0.14-0.25 Gy might significantly increase the risk for astronauts. Further work will be needed to confirm that this extrapolation from mice to humans is valid. Although 56Fe targeted to the aortic arch accelerated development of atherosclerosis, the dose dependence of 56Fe-induced atherosclerosis varied by site. While 5 Gy was required for a significant effect in the aortic arch, 2 Gy was sufficient in the aortic root (where the aorta emerges from the heart) and carotid arteries. These differences might be due to pro-atherogenic contributions of local factors, such as shear stress (the force felt by the inside wall of the vessel from blood flowing by). This advances our understanding of the how radiation induces atherosclerosis by demonstrating that, while direct radiation effects on the vessel are clearly a component of the 56Fe effect on atherosclerosis, the risk can be modified by other local factors.

The above experiments were done by irradiating only the heart and major blood vessels of mice. Since the entire astronaut will be irradiated during spaceflight, we then asked the question of whether radiation-induced atherosclerosis is primarily dependent on local effects on the vessels themselves or whether significant contributions, either positive or negative, from other organ systems might be important when the whole body is exposed. At least a component of the pro-atherogenic effect of 56Fe was a direct effect on the vessel itself, because radiation did not increase atherosclerosis in un-irradiated areas of the aorta. However, potential effects on the immune system may either exacerbate or ameliorate the local radiation effects. Therefore, we compared whole-body irradiation to that targeted to the chest and neck. Our data indicate that, as with direct, local factors, the influence of systemic factors also depends on the site and perhaps on the particular effect measured. Thickening of the carotid artery wall, a hallmark of atherosclerosis, was largely unaffected by 56Fe irradiation of the rest of the body. Plaque formation in the aortic arch, however, seemed to be slightly blunted in whole-body 56Fe irradiated mice vs. those with radiation targeted to the major vessels. This was a minor effect however, because significant exacerbation of plaques was seen at 2 Gy in either case. We conclude that the major effect of radiation is on the vessels themselves, but that systemic effects from irradiation of other tissues may modify the damage to a small extent.

Degen-2, “What are the mechanisms of degenerative tissues risks in the heart, circulatory, endocrine, digestive, lens, and other tissue systems? What surrogate endpoints do they suggest?” The cell biology arm of this study (Aim #2) is specifically designed to elucidate the mechanism of radiation-induced atherosclerosis. It is known that gamma- and x-radiation increase the adhesiveness of vascular endothelium, a key, early step in the pathogenesis of atherosclerosis. We established that 56Fe increases adhesiveness as well. It is hypothesized that this increased endothelial adhesiveness initiates endovascular inflammation in atherosclerosis-prone arteries, and that this inflammatory response can become self-perpetuating. Although a common mechanism for making cells stick to each other is to increase the number of adhesion molecules on the surface of the cell, we showed that x-ray-induced endothelial adhesiveness can occur even without up-regulation of adhesion molecules. We further showed that the mechanism by which this occurs is dependent upon chemokine signaling. We determined that the integrin alpha4beta1 (also known as VLA-4), an important adhesion molecule needed for atherosclerosis to develop, is one of the adhesion molecules activated by radiation. This was not a direct effect, but was mediated by production of signaling molecules, known as chemokines, by cells in the blood vessels.

Experiments at Brookhaven National Laboratory (where particle accelerators can mimic components of cosmic radiation) then confimed that, despite the major differences in how x-rays and heavy ions interact with tissue, 56Fe also increases endothelial adhesiveness through a chemokine-dependent mechanism. Moreover, the 56Fe radiation-induced adhesiveness also depends on signaling to the integrin alpha4beta1.

Degen-3, “What are the progression rates and latency periods for degenerative risks, and how do progression rates depend on age, gender, radiation type, or other physiological or environmental factors?”

Degen-3 is addressed by Aims #1 and #3. Since Aim #1 involves experiments with both x-rays and 56Fe, progression rates for these two radiation types can be compared. We showed earlier that x-rays and 56Fe have similar pro-atherogenic effects, increasing adhesiveness of human aortic endothelial cells. The dose of 56Fe required to do this was much lower than that required for x-rays, however.

We also demonstrated a linear energy transfer (LET) dependence of radiation-induced atherosclerotic changes in the major blood vessels. Thus, to estimate risk for astronauts on deep space missions it might be better to consider not only the total dose of heavy ion likely to be absorbed, but to take into account the mix of ions and their energies as well.

NOTE: End date changed to 7/31/2014 per NSSC information (Ed., 4/10/14)

NOTE: End date changed to 4/30/2014 per NSSC information (Ed., 1/22/14)

In our last project, we established that X-ray, 56Fe (iron ion), and proton irradiation of blood vessel cells increase adhesiveness of the vessel wall, and that X-rays and 56Fe accelerate development of atherosclerosis in a mouse model (results of proton experiments are pending). The molecular mechanism for this, however, is not yet known. In addition, it remains to be determined how fractionation of doses and irradiation of other tissues affect the dose dependence of both cell adhesion and development of atherosclerosis.

With the hypothesis that radiation in general and cosmic radiation in particular directly alter the adhesive properties of vascular endothelium, and resultant vascular inflammation accelerates atherosclerosis, we propose to systematically investigate mechanisms of radiation effects on vascular cells, using both isolated cells and whole mice, to better predict risk and to provide the basis to develop possible future countermeasures. Our specific aims are:

Aim 1: Determine whether atherogenic effects of radiation are limited to local effects on vascular endothelium, or if other systems contribute to disease progression and/or modify dose dependence.

Aim 2: Determine the molecular mechanism of acute activation of leukocyte-endothelial cell adhesion in response to radiation.

Aim 3: Determine how fractionation of doses affects dose-dependence of progression rates, latency periods, and surrogate endpoints.

The risk from accidental exposure is similar. For example, atomic bomb survivors have an increased incidence of coronary artery disease and stroke. Risk for cardiovascular disease after radiation exposure at Chernobyl was increased for those who were exposed to less than 1 Gy. Even radiation technologists in the 1950s (when shielding was less rigorous) had an increased risk of death from cardiovascular disease, demonstrating that repeated exposure at low doses results in significant risk years later. Currently, the principal strategy for reducing risk is avoidance of exposure.

Completion of our specific aims will advance the knowledge of the molecular mechanisms of radiation-induced atherosclerosis, enabling better prediction of cardiovascular risk from exposure, facilitating early detection through the use of surrogate biomarkers, and pointing the way toward potential countermeasures to mitigate the cardiovascular consequences of radiation exposure, both in space and on Earth.

Progress on each aim is summarized below.

Aim 1: Determine whether atherogenic effects of radiation are limited to local effects on vascular endothelium, or if other systems contribute to disease progression and/or modify dose dependence. We performed studies examining 56Fe-induced thickening of the wall of the carotid artery for both radiation targeted to the chest and neck and radiation of the whole mouse. Ten week old male ApoE -/- mice (a well-characterized animal model of atherosclerosis) were anesthetized by injection of the drugs ketamine and xylazine and irradiated with either 0 Gy , 2.0 Gy , or 5 Gy 56Fe (accelerated iron ions). The mice were returned to our home institution and fed a normal mouse-chow diet and housed under standard conditions. At 13 weeks post-irradiation (when the mice were 23 weeks of age), mice were euthanized and dissected. Increased thickness of the intimal layer of the wall of the carotid artery, an atherosclerotic change, was compared between mice that received radiation only to the chest and neck and those that received radiation to the entire body. In addition, atherosclerotic changes in the aortic root (where the aorta connects to the heart) were also examined. Results of this study are now complete and are being prepared for publication.

Aim 2: Determine the molecular mechanism of acute activation of leukocyte-endothelial cell adhesion in response to radiation. We showed previously that both x-irradiation and 56Fe increase the adhesiveness of vascular endothelial cells (the cells that line the inside of the vessel wall), an important, early step in the development of atherosclerosis. We also demonstrated that this effect depends on chemokines, a family of signaling molecules that is involved in both cell adhesion and atherosclerosis. In the past year, we worked to identify the particular chemokine responsible. For this study, endothelial cells from human aortas were grown in dishes to simulate the lining of the aorta.

First, cell-growth media, the liquid that nourishes cells grown in dishes, was collected from the human aortic endothelial cells (HAECs) 24 hours after irradiation from both x-irradiated and sham-irradiated control cells (cells that were manipulated exactly as the irradiated cells, but not actually irradiated). A Luminex assay was then used to determine the concentration of several chemokines, focusing on those that were likely to be secreted by endothelial cells and known to be involved in atherosclerosis. From this, we could determine which chemokine(s) were secreted in response to radiation.

When candidate chemokines were identified, we tested for their importance in the mechanism of adhesiveness changes by inhibiting their action using specific antibodies. This was to determine how necessary the candidate chemokines are for the increase in adhesion. We also tested whether each chemokine was sufficient by itself to reproduce the radiation effect. That is, we added individual chemokines to un-irradiated cells to determine whether they could increase adhesiveness by themselves. This study is also complete and is being prepared for publication.

Another important question concerning the mechanism of radiation effects on atherosclerosis is how different types of radiation affect the disease process. This is very important, because cosmic radiation is very different from the x-rays used in diagnostic medicine (that is, the x-rays and CT scans used to see inside the body) and in cancer treatments. Most of what we know about radiation effects on atherosclerosis comes from studies of such terrestrial radiation sources. In space, however, astronauts will be exposed to accelerated ions as well as x-rays, and these ions interact with human tissue very differently than do x-rays. An understanding of the similarities and differences between different types of radiation with respect to the arterial damage they cause will be essential to enable us to predict risk to astronauts. The knowledge gained will also be important for understanding new types of radiation therapy, such as proton therapy, that patients are now receiving in leading hospitals in the USA and abroad.

To address this question, we irradiated mice with x-rays or either 56Fe or 28Si, two different ions found in cosmic radiation. We used the same 10-week old ApoE -/- mouse model that we had used in previous studies. Again, we waited 13 weeks after radiation to assess atherosclerosis-related radiation effects.

When the mice were 23 weeks old, they were euthanized and atherosclerotic changes were examined in the carotid artery, the aortic root, and in other parts of the aorta. We have now determined that there are both similarities and differences in the effects of these three types of radiation. The results are to be presented and their consequences discussed in an upcoming publication, now in preparation.

Aim 3: Determine how fractionation of doses affects dose-dependence of progression rates, latency periods, and surrogate endpoints. We also compared 10-week old male mice that were either un-irradiated, irradiated with a single dose of 2 Gy iron ions on one day, or irradiated with 5 doses of 0.4 Gy iron ions on each of 5 consecutive days. The experiments are finished, but the data are still being analyzed. Results will be included in the final progress report to be written after the conclusion of this funding period on July 31, 2015.

NOTE: End date changed to 7/31/2014 per NSSC information (Ed., 4/10/14)

NOTE: End date changed to 4/30/2014 per NSSC information (Ed., 1/22/14)

In our last project, we established that X-ray, 56Fe (iron ion) and proton irradiation of blood vessel cells increase adhesiveness of the vessel wall, and that X-rays and 56Fe accelerate development of atherosclerosis in a mouse model (results of proton experiments are pending). The molecular mechanism for this, however, is not yet known. In addition, it remains to be determined how fractionation of doses and irradiation of other tissues affect the dose dependence of both cell adhesion and development of atherosclerosis.

With the hypothesis that radiation in general and cosmic radiation in particular directly alter the adhesive properties of vascular endothelium, and resultant vascular inflammation accelerates atherosclerosis, we propose to systematically investigate mechanisms of radiation effects on vascular cells, using both isolated cells and whole mice, to better predict risk and to provide the basis to develop possible future countermeasures. Our specific aims are:

Aim 1: Determine whether atherogenic effects of radiation are limited to local effects on vascular endothelium, or if other systems contribute to disease progression and/or modify dose dependence.

Aim 2: Determine the molecular mechanism of acute activation of leukocyte-endothelial cell adhesion in response to radiation.

Aim 3: Determine how fractionation of doses affects dose-dependence of progression rates, latency periods, and surrogate endpoints.

The risk from accidental exposure is similar. For example, atomic bomb survivors have an increased incidence of coronary artery disease and stroke. Risk for cardiovascular disease after radiation exposure at Chernobyl was increased for those who were exposed to less than 1 Gy. Even radiation technologists in the 1950s (when shielding was less rigorous) had an increased risk of death from cardiovascular disease, demonstrating that repeated exposure at low doses results in significant risk years later. Currently, the principal strategy for reducing risk is avoidance of exposure.

Completion of our specific aims will advance the knowledge of the molecular mechanisms of radiation-induced atherosclerosis, enabling better prediction of cardiovascular risk from exposure, facilitating early detection through the use of surrogate biomarkers, and pointing the way toward potential countermeasures to mitigate the cardiovascular consequences of radiation exposure, both in space and on Earth.

Aim 2: Determine the molecular mechanism whereby radiation leads to activation of leukocyte-EC adhesion. Radiation increases the adhesiveness of vascular endothelial cells, an important, early step in the development of atherosclerosis. This year, we published results demonstrating that x-ray induced endothelial adhesiveness can occur even without a change in the expression level of pro-atherogenic adhesion molecules. That is, even though endothelial cell adhesiveness was increased by radiation, cell surface expression of key endothelial adhesion molecules did not significantly increase. We then showed that the increased adhesiveness was due to signaling by the endothelial cells to the leukocytes, activating receptors on the leukocytes to increase adhesion between the two cell types. We have now completed experiments demonstrating that iron ion radiation, an important component of cosmic radiation that is very different from x-rays, also increases endothelial cell adhesiveness by a chemokine-dependent mechanism. Studies are underway to further clarify the similarities and differences between x-ray and iron ion effects to determine whether other knowledge about the mechanism of action of x-rays can also be used to understand the potentially pro-atherogenic effects of cosmic radiation.

Aim 3: Determine how fractionation of doses affects dose-dependence of progression rates, latency periods, and surrogate endpoints. In November, 2012, we performed an experiment in which 10-week old male mice were either un-irradiated, irradiated with a single dose of 2 Gy iron ions, or irradiated with 5 doses of 0.4 Gy iron ions each on 5 consecutive days. These mice will be raised under standard conditions and fed a normal diet until they are analyzed at 13 weeks post-irradiation.

In our last project, we established that X-ray, 56Fe (iron ion) and proton irradiation of blood vessel cells increase adhesiveness of the vessel wall, and that X-rays and 56Fe accelerate development of atherosclerosis in a mouse model (results of proton experiments are pending). The molecular mechanism for this, however, is not yet known. In addition, it remains to be determined how fractionation of doses and irradiation of other tissues affect the dose dependence of both cell adhesion and development of atherosclerosis.

With the hypothesis that radiation in general and cosmic radiation in particular directly alter the adhesive properties of vascular endothelium, and resultant vascular inflammation accelerates atherosclerosis, we propose to systematically investigate mechanisms of radiation effects on vascular cells, using both isolated cells and whole mice, to better predict risk and to provide the basis to develop possible future countermeasures. Our specific aims are:

Aim 1: Determine whether atherogenic effects of radiation are limited to local effects on vascular endothelium, or if other systems contribute to disease progression and/or modify dose dependence.

Aim 2: Determine the molecular mechanism of acute activation of leukocyte-endothelial cell adhesion in response to radiation.

Aim 3: Determine how fractionation of doses affects dose-dependence of progression rates, latency periods, and surrogate endpoints.

The risk from accidental exposure is similar. For example, atomic bomb survivors have an increased incidence of coronary artery disease and stroke. Risk for cardiovascular disease after radiation exposure at Chernobyl was increased for those who were exposed to less than 1 Gy. Even radiation technologists in the 1950s (when shielding was less rigorous) had an increased risk of death from cardiovascular disease, demonstrating that repeated exposure at low doses results in significant risk years later. Currently, the principal strategy for reducing risk is avoidance of exposure.

Completion of our specific aims will advance the knowledge of the molecular mechanisms of radiation-induced atherosclerosis, enabling better prediction of cardiovascular risk from exposure, facilitating early detection through the use of surrogate biomarkers, and pointing the way toward potential countermeasures to mitigate the cardiovascular consequences of radiation exposure, both in space and on Earth.

We had established in our last project that 2-5 Gy 56Fe targeted to the upper aorta and the carotid arteries of 10-week old apoE -/- mice accelerated the development of atherosclerosis by 23 weeks of age. This radiation dose is 4-8 times lower than the X-ray dose required to produce the same effect in this well-characterized mouse atherosclerosis model. Atherosclerosis was exacerbated in irradiated portions of the aorta, but not in un-irradiated portions of the same vessel, indicating that at least part of the mechanism for radiation-induced atherosclerosis is a direct effect on the vessels. The purpose of targeting the radiation was to exclude effects on other organs and on the immune system which might contribute to the disease process and cloud interpretation of direct effects. However, since astronauts will receive whole-body 56Fe irradiation on interplanetary missions, it is important now to take this to the next level of complexity and determine whether effects on extravascular systems also contribute to atherosclerosis progression. To address this question, 10-week old apoE -/- mice were exposed to 2-5 Gy full body 56Fe irradiation in May, 2011 and were dissected 13 weeks later. Aortic en face preparations and aortic root and carotid artery cross sections were prepared for histologic analysis as had been done previously for targeted radiation experiments. Analysis is currently being performed to determine whether disease progression in the aortic arch and carotid arteries is modified by irradiation of the remainder of the animal.

Aim 2:Determine the molecular mechanism whereby radiation leads to activation of leukocyte-EC adhesion.

This year, we published results demonstrating that radiation-induced adhesiveness is not due to increased expression of adhesion molecules, but is a result of chemokine-dependent signaling from the endothelial cell to the leukocyte. That is, even though endothelial cell adhesiveness was increased, cell surface expression of key endothelial adhesion molecules, including ICAM-1, VCAM-1, E-selectin, and P-selectin, did not significantly increase following irradiation (as measured by flow cytometry). Blocking the leukocyte receptors for ICAM-1 and VCAM-1, however, abrogated the radiation-induced adhesiveness. Since these receptors are integrins, a group of adhesion molecules that exist in multiple activation states, we checked whether integrin activation played a role in radiation-induced adhesion. Pre-treatment of the leukocytes with pertussis toxin, which blocks chemokine-dependent integrin activation, blocked the increased endothelial cell-leukocyte adhesion. Since the endothelial cells were irradiated, but the leukocytes were not, this suggests that radiation stimulated chemokine signaling by the endothelial cells to the leukocytes, activating integrins on the leukocytes to increase adhesion between the two cell types.

This year, we have identified several endothelial cell-expressed chemokines that seem to be involved in this mechanism. Experiments are currently underway to determine the relative importance of trans-membrane chemokines, anchored in the cell membrane, and those that are secreted to bind to carbohydrates on the cell surface.

Aim 3: Determine how fractionation of doses affects dose-dependence of progression rates, latency periods, and surrogate endpoints.

Awaiting identification of surrogate endpoints.

In our current project, we established that X-ray, 56Fe (iron ion) and proton irradiation of blood vessel cells increase adhesiveness of the vessel wall, and that X-rays and 56Fe accelerate development of atherosclerosis in a mouse model (results of proton experiments are pending). The molecular mechanism for this, however, is not yet known. In addition, it remains to be determined how fractionation of doses and irradiation of other tissues affect the dose dependence of both cell adhesion and development of atherosclerosis.

With the hypothesis that radiation in general and cosmic radiation in particular directly alter the adhesive properties of vascular endothelium, and resultant vascular inflammation accelerates atherosclerosis, we propose to systematically investigate mechanisms of radiation effects on vascular cells, using both isolated cells and whole mice, to better predict risk and to provide the basis to develop possible future countermeasures. Our specific aims are:

Aim 1: Determine whether atherogenic effects of radiation are limited to local effects on vascular endothelium, or if other systems contribute to disease progression and/or modify dose dependence.

Aim 2: Determine the molecular mechanism of acute activation of leukocyte-endothelial cell adhesion in response to radiation.

Aim 3: Determine how fractionation of doses affects dose-dependence of progression rates, latency periods, and surrogate endpoints.